Organoboron compounds are useful for a variety of biomedical applications (see, e.g., Das, et al. Future Med. Chem. 2013, 5, 653.). For instance, 10B-containing molecules are widely used in boron neutron capture therapy (BNCT), a radiation therapy for the treatment of malignant tumors (see, e.g., Barth, et al. Clin. Cancer Res. 2005, 11, 3987). In addition, there is interest of organoboron compounds to treat diseases, as exemplified by bortezomib (Velcade), the first FDA-approved boron-based drug for the treatment of multiple myeloma and mantle cell lymphoma (see, e.g., Gorovoy, et al. Chem. Biol. Drug Des. 2013, 81, 408; Richardson, et al. Annu. Rev. Med. 2006; Vol. 57, p 33; Smoum, et al. Chem. Rev. 2012, 112, 4156.). The prevalence of organoborons necessitates the development of new methods for preparing molecules with high efficiency and selectivity. Carbenoid insertion into a B-H bond represents a very appealing strategy for introducing boron atoms into organic molecules that offers several distinct advantages over other borylation methods. It utilizes tetravalent boranes as borane reagents, which are much more stable, less toxic, and easier to handle than the Lewis-acidic trivalent borane reagents used in most borylation reactions (see, e.g., Curran, et al. Angew. Chem. Int. Ed. 2011, 50, 10294). Furthermore, carbenoid B-H insertion is highly desirable for the synthesis of α-borylcarbonyl molecules, which can be easily derivatized to a wide range of functionalized organoborons such as β-boryl alcohols, enamines, and enol ethers (see, e.g., He, et al. Acc. Chem. Res. 2014, 47, 1029; He, et al. Dalton Trans. 2014, 43, 11434; He, et al. J. Am. Chem. Soc. 2011, 133, 13770; He, et al. J. Am. Chem. Soc. 2012, 134, 9926.). Examples of metal-catalyzed carbene insertions into B-H bonds were reported only very recently with rhodium and copper-based catalysts (see, e.g., Li, et al. J. Am. Chem. Soc. 2013, 135, 12076; Cheng, et al. J. Am. Chem. Soc. 2013, 135, 14094; Chen, et al. J. Am. Chem. Soc. 2015, 137, 5268.). These reactions allow the preparation of α-borylcarbonyl compounds, but not without limitations. In addition to low catalyst turnovers (<100) and hazardous halogenated solvents, these reactions are narrow in scope: only α-aryl-α-diazocarbonyl compounds are accommodated in the enantioselective B-H insertion reactions reported in the literature.
Enzymes are remarkable for catalyzing chemical transformations with high chemo-, regio-, and enantio-selectivity under environmentally benign conditions (see, e.g., Bornscheuer, et al. Nature 2012, 485, 185; Bloom, Arnold. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 9995; Reetz, J. Am. Chem. Soc. 2013, 135, 12480.). Therefore, biocatalysis has become increasingly important for the pharmaceutical industry, with applications ranging from drug discovery to industrial-scale pharmaceutical production (see, e.g., Tucker, et al. Nature 2016, 534, 27; Tao, et al. Biocatalysis for the Pharmaceutical Industry: Discovery, Development, and Manufacturing; Wiley, 2009.). Despite the myriad of currently available biocatalysts, however, enzymes that catalyze the formation of carbon-boron (C—B) bonds are unknown. During the past four years, the Arnold laboratory has developed a biocatalytic platform that uses cytochrome P450s, nature's most versatile oxidation catalysts, for a broad range of non-natural transformations including alkene cyclopropanation, aziridination, aliphatic C—H amination, sulfimidation, and carbenoid insertion into N—H bonds (see, e.g., Hernandez, Arnold, et al. ACS Catalysis 2016, 6, 7810; Renata, Arnold, et al. Angew. Chem. Int. Ed. 2015, 54, 3351; Wang, Arnold, et al. Chem. Sci. 2014, 5, 598; Hyster, Arnold. lsr. J. Chem. 2015, 55, 14; Coelho, Arnold, et al. Nat. Chem. Biol. 2013, 9, 485; Arnold. Q. Rev. Biophys. 2015, 48, 404; Coelho, Arnold, et al. Science 2013, 339, 307; Kan, Arnold, et al. Science 2016, 354, 1048.). They and others have also demonstrated that other heme-containing proteins can catalyze carbenoid insertion and other reactions (see also, e.g., Bordeaux, et al. Bioorg. Med. Chem. 2014, 22, 5697; Bordeaux et al. Angew. Chem. Int. Ed. 2015, 54, 1744; Sreenilayam, et al. Chem. Commun. 2015, 51, 1532; Tyagi, et al. Chem Sci. 2015, 6, 2488; Bajaj, et al. Angew. Chem. Int. Ed. 2016, 55, 16110; Tyagi, et al. Angew. Chem. Int. Ed. 2016, 55, 2512; Giavani, et al. Chem Sci. 2016, 7, 234; Gober, et al. ChemBioChem 2016, 17, 394.). However, C—B bond formation using an enzyme has never been demonstrated. Harnessing the highly evolvable nature of hemoproteins toward metal-carbenoid chemistries, herein we developed the first biocatalytic C—B bond forming transformation. We have used directed evolution of hemoproteins to create enzymes that effect iron-carbenoid insertion into boron-hydrogen bonds for the first time.
Recent advances in enzyme engineering and design have expanded nature's catalytic repertoire to functions that are new to biology1-3. Yet only a subset of these engineered enzymes can function in living systems4-7. Finding enzymatic pathways that forge chemical bonds not found in biology is particularly difficult in the cellular environment, as this hinges on the discovery not only of new enzyme activities but also reagents that are simultaneously sufficiently reactive for the desired transformation and stable in vivo. Here we report the discovery, evolution, and generalisation of a fully genetically-encoded platform for producing chiral organoboranes in bacteria. Escherichia coli harbouring wild-type cytochrome c from Rhodothermus marinus8 (Rma cyt c) were found to form carbon-boron bonds in the presence of borane-Lewis base complexes, through carbene insertion into B—H bonds. Directed evolution of Rma cyt c in the bacterial catalyst provided access to 16 novel chiral organoboranes. The catalyst is suitable for gram scale biosynthesis, offering up to 15300 turnovers, 6100 h−1 turnover frequency, 99:1 enantiomeric ratio (e.r.), and 100% chemoselectivity. The enantio-preference of the biocatalyst could also be switched to provide either enantiomer of the organoborane products. Evolved in the context of whole-cell catalysts, the proteins were more active in the whole-cell system than in purified forms. This study establishes a DNA-encoded and readily engineered bacterial platform for borylation; engineering can be accomplished at a pace which rivals the development of chemical synthetic methods, with the ability to achieve turnovers that are two orders of magnitude (over 400-fold) greater than that of known chiral catalysts for the same class of transformation9-11. This tunable method for manipulating boron in cells opens a whole new world of boron chemistry in living systems.
Boron-containing natural products are synthesised in the soil by the myxobacterium Sorangium cellulosum as antibiotics against Gram-positive bacteria12. In the sea, these molecules give the Jurassic red alga Solenopora jurassica its distinct pink colouration13; they are also produced by the bioluminescent bacterium Vibrio harveyi for cell-cell communications14 (FIG. 1). To prepare boron-containing biomolecules, living organisms produce small molecules that spontaneously react with boric acid available in the environment15,16. While this non-enzymatic method for capturing boron is sufficient for an organism's survival, it is limited to a substrate's inherent affinity towards boric acid, and lacks tunability and generality for synthetic biology applications. Moreover, organisms that produce organoboranes (compounds that contain carbon-boron bonds) are unknown.